During aging, an increasing proportion of total lens proteins becomes water insoluble (WI) either due to aggregation and/or cross-linking. The increased sizes of cross-linked multimers of crystallins become so large that they finally become water insoluble and cause lens opacity during age-related (senile) cataract development. Among the variety of post-translational modifications, deamidation and truncations of crystallins are identified as the most abundant during aging in human lenses. Therefore, these modifications playa major role in age-related aggregation and cross-linking of crystallins, and in tum, are significant causative factors in age-related cataract development. Our studies have shown that ~A3-crystallin exists as an activable proteinase in the lens, and the active enzyme is capable of proteolyzing aA-, aB-, yC- and yD-crystallins. Further, our studies demonstrated that ~A3 proteinase is inhibition by aA- and aB-crystallins. Based on these results, we have hypothesized that ~A3-proteinase activity is regulated in vivo by aA- and aB-crystallins as inhibitors, and the activated ~A3-proteinase proteolyzes a-, ~- and y-crystallins. The crystallin fragments per se aggregate and/or undergo post-translational modifications such as deamidation. The unmodified and modified crystallin fragments aggregate and cross-link with intact crystallins to first form the water soluble-high molecular weight (WS-HMW) proteins, where its components cross-link and become water insoluble. To test the above hypothesis, the proposed studies will be focused to answer the following two questions: (1) Which polypeptide (amino acids) forms the ~A3 proteinase active site, and how is the proteinase activity inhibited by aA- and aB-crystallins? (2) What are the roles of crystallin fragments and/or deamidated crystallins in aggregation and cross-linking processes of crystallins in vivo? To answer the first question, we will determine the ~A3-proteinase active site in the regions of the motifs III and IV, the proteinase-induced proteolysis of a-, ~- and y-crystallins in vivo, and the inhibition mechanism of ~A3-proteinase by aA and aB-crystallins. To answer the second question, we will determine whether the fragments of a-, ~- and y-crystallins are post-translationally modified in vivo during aging and cataract development, the mechanism of complex formation between crystallin fragments and deamidated crystallins, and effects of deamidation of Asn(s) in aA- and aB-crystallins on lens transparency using transgenic mouse models. Because human lenses will be used in these studies, the findings will be relevant in elucidation of in vivo properties of ~A3-proteinase, its regulation by aA- and aB-crystallins as inhibitors, the ~A3- proteinase-induced proteolysis of crystallins, and potential roles of protelyzed crystallin fragments and their deamidated species in aggregation and cross-linking process during development of opacity in aging human lenses. PHS